Changes in SARS-CoV-2 seroprevalence and population immunity in Finland, 2020–2022

ABSTRACT Studying the prevalence of SARS-CoV-2 specific antibodies (seroprevalence) allows for assessing the impact of epidemic containment measures and vaccinations and estimating the number of infections regardless of viral testing. We assessed antibody-mediated immunity to SARS-CoV-2 induced by infections and vaccinations from April 2020 to December 2022 in Finland by measuring serum IgG to SARS-CoV-2 nucleoprotein (N-IgG) and spike glycoprotein from randomly selected 18–85-year-old subjects (n = 9794). N-IgG seroprevalence remained at <7% until the last quartile (Q) of 2021. After the emergence of the Omicron variant, N-IgG seroprevalence increased rapidly and was 31% in Q1/2022 and 54% in Q4/2022. Seroprevalence was highest in the youngest age groups from Q2/2022 onwards. We did not observe regional differences in seroprevalence in 2022. We estimated that 51% of the Finnish 18–85-year-old population had antibody-mediated hybrid immunity induced by a combination of vaccinations and infections by the end of 2022. In conclusion, major shifts in the COVID-19 pandemic and resulting population immunity could be observed by serological testing.

During the early stages of the COVID-19 epidemic in Finland, access to viral testing was very limited [6,7], and other methods were needed to estimate the incidence of SARS-CoV-2 infections. The serological population study of the coronavirus epidemic in Finland [8] was first initiated in early 2020 to assess how widespread SARS-CoV-2 infections were in the population and how many had developed antibodies against the virus. Later the continuous monitoring of seroprevalence (serosurveillance) was also used to assess the impact of containment measures, emerging variants and the effectiveness of vaccination programmes.

COVID-19 vaccinations began in Finland in
December 2020, with healthcare workers, the elderly, and those at the highest risk of severe COVID-19 being the first to whom vaccines were offered [9]. Vaccinations were gradually extended so that by July 2021 all 18-year-olds and older were eligible. Although PCR testing capacity caught on and was widely accessible from August 2020 in Finland [7,10], it remained important to follow population immunity and assess whether viral testing captured most infections. In late-2021 Finland changed to less comprehensive PCR testing [11,12] due to the availability of the athome antigen tests and the rapid increase in infections caused by the Omicron variant [13,14]. However, athome antigen test results were not reported to any register, and serology-based estimations on the number of SARS-CoV-2 infections became indispensable again.
Monitoring the gradual development of the population immunity elicited by SARS-CoV-2 infections and/or vaccinations has been crucial in the planning of epidemic containment measures and vaccination programme in Finland. Here, we present the results of a continuing serosurveillance study spanning from April 2020 to December 2022, including the effects of Omicron emergence on the Finnish adult population seroprevalence and hybrid immunity.

Study design and population
We invited 34,619 subjects by regular mail to participate in the study in forty sequential random population surveys between April 2020 and December 2022. The size of each population survey was adapted to the expected true seroprevalence in the population to achieve a predefined 2-3 percentage point accuracy in the 90% interval estimate of seroprevalence. Due to the properties of the binomial distribution, the required sample size is highest when the true seroprevalence is close to 50%, and lowest when it is close to 0% or 100%. Initially, the target size of each survey was 750, which is sufficient for 2 percentage point accuracy in the estimate in a population where seroprevalence is low (<10%). As seroprevalence estimates remained below 5%, the size of the surveys was lowered (Table S1). As the seroprevalence was expected to have increased by 2022 due to the emergence of more infectious variants, the size of the sample was increased accordingly, with a target size of 1300 per survey. This latter sample size is sufficient for 2 percentage point accuracy when the prevalence is below 20%, and 3 percentage point accuracy when prevalence is 50%. Note that in the current study, we report 95% confidence intervals for seroprevalence.
During 2020-2021 invitations were sent to 18-70year-olds and in 2022 to 18-85-year-olds. Individuals living within institutional care and those previously invited to this study or the follow-up study of the serological population survey of the coronavirus epidemic [15] were excluded. We asked the participants to provide a blood sample for antibody testing at their local healthcare district laboratory. We retrieved information on COVID-19 vaccinations and PCRconfirmed SARS-CoV-2 infections from Finland's national vaccination register and infectious diseases register utilizing the Finnish personal identity code for data linkage. The subjects who participated in 2022 were also asked if they had had a positive athome antigen test and if yes, when.
We also retrieved Finnish adult population key metrics from The Finnish Population Information System for comparison to the study population. We extracted the number of COVID-19 cases in 18-85year-olds in 2020-2022 from the infectious diseases register and converted it to cumulative incidence per quartile. The number of first COVID-19 vaccine doses given to over 18-year-olds were extracted from THL's User Interface for Database Cubes and Reports [16] per week and converted it to cumulative coverage per quartile.

SARS-CoV-2 fluorescent multiplex immunoassay
We measured the concentration of serum IgG antibodies to SARS-CoV-2 nucleoprotein (N-IgG) and two spike glycoprotein (S-IgG) antigens with an inhouse fluorescent multiplex immunoassay (FMIA) and interpreted the measurements as positive or negative as previously described [17] with slight modifications detailed in the supplementary material. The IgG SARS-CoV-2 FMIA is an accredited assay at the Expert Microbiology Unit of the Finnish Institute of Health and Welfare, which is a testing laboratory T077 accredited by FINAS Finnish Accreditation Service, accreditation requirement SFS-EN ISO/IEC 17025. The IgG SARS-CoV-2 FMIA assay has been calibrated to the WHO international standard [18].

Definitions for infection-induced and vaccineinduced immunity
We assessed the quality of antibody-mediated immunity by combining N-IgG and S-IgG antibody results with COVID-19 vaccination status and separated subjects into five non-overlapping categories of immunity: no immunity, infection immunity, vaccination immunity, vaccinated without response, hybrid immunity (Table 1).

Statistical methods
We present results separately for the proportion of individuals with N-IgG and S-IgG antibodies (N-IgG seroprevalence, S-IgG seroprevalence). Serum samples were grouped by sample collection date into year quartiles (Q), with samples collected from January to March comprising Q1, April to June Q2, July to September Q3 and October to December Q4. We estimated seroprevalence separately for each timepoint both within and across age groups (18-29, 30-44, 45-64 and 65-85 years), and report point estimates and 95% confidence intervals. For estimation within age groups, we used the Clopper-Pearson method for confidence intervals. For estimation across age groups, we adjusted for differences in the sample population and our target population (those 18-85years-old living in Finland) by weighting each age group's seroprevalence estimate by the age group's Hybrid immunity Yes Yes Yes population proportion. The age-adjusted estimates and their confidence intervals were based on logistic regression and a Wald-type interval estimate constructed on the log-odds scale, as implemented in the survey R package [19].

Informed consent and human experimentation guidelines
Participation was voluntary. The study protocol was approved by the ethical committee of the Hospital District of Helsinki and Uusimaa and registered under the study protocol HUS/1137/2020. Written informed consent was obtained from all study subjects before sample collection.

Description of the study population
Of the 34,619 invited subjects, 9794 (28%) donated sera from April 2020 to December 2022 in the five largest healthcare districts in Finland, i.e. Helsinki and Uusimaa, Pirkanmaa, Northern Ostrobothnia, Northern Savonia and Southwest Finland. Participation decreased during the study from 64% in the first sample in 2020 to 19% in the last sample of 2022 (Table S1). The distribution of sample collection per month and year is presented in Table S2. Participant demographics and comparison to the adult population in Finland and the capital region (Uusimaa) are summarized in Table  S3. More females (61%) than males (39%) donated sera. Compared to Finland's nationwide age and native language distributions, 18-29-year-olds participated less frequently, whilst the 45-64-year-old and native Finnish or Swedish speaker groups were disproportionally large (Table S3). Additionally, 96% of the participants had received at least one COVID-19 vaccine before 2022, exceeding the 88% vaccination rate of 18-85-year-olds in Finland during the same period (Table S3). Most samples (54%) were collected in Finland's capital region (Helsinki and Uusimaa healthcare district). Regional distributions varied with random population surveys (Table S1, Table S4).

Assay sensitivity in detection of past SARS-CoV-2 infection
For samples collected 0-5 months, 6-12 months and over 12 months after PCR-confirmed infection, 90% (n = 291), 74% (n = 146) and 65% (n = 26) had N-IgG, respectively. From March 2022 onwards participants were asked if they had received a positive at-home antigen test result. When we combined the N-IgG results of those with a positive PCR and/or at-home antigen test, the estimates remained similar. The N-IgG assay sensitivity in the detection of past SARS-CoV-2 infection confirmed by either PCR or at-home antigen test was 91% at 0-5 months (n = 641), 74% at 6-12 months (n = 292) and 62% at over 12 months (n = 21) after the most recent positive test. S-IgG assay sensitivity estimated from unvaccinated subjects was 82% (n = 62) for samples taken up to 12 months after infection. S-IgG assay sensitivity could not be assessed for samples taken over a year after infection (n = 2). S-IgG sensitivity in the detection of antibodies induced by vaccination alone or vaccination and infection was 99% (n = 3288) including all samples from subjects with at least one dose ≥14 days before sample collection, the last of which was collected 559 days after the most recent COVID-19 vaccine dose. We were unable to assess the effect of possible reinfections as we did not have repeated samples from the subjects.

Regional differences in seroprevalence
Until Q1/2021 seroprevalences were similar or higher in Helsinki and Uusimaa compared to other areas combined ( Figure S1). During that time, differences in the seroprevalence estimates were larger for S-IgG than for N-IgG, and for both, the interval estimates were overlapping. The N-IgG seroprevalence was slightly higher in Helsinki and Uusimaa compared to other areas in Q2/2021. During Q3/2021-Q1/2022 there were no sufficient data for estimating seroprevalence in areas other than Helsinki and Uusimaa. In 2022, there were no clear differences in seroprevalence  between the areas, but during Q4/2022 the N-IgG seroprevalence was slightly higher in areas other than Helsinki and Uusimaa, however with overlapping interval estimates between the areas ( Figure S1).

Test-confirmed infections in 2020-2022
In Q2/2020 under 15% of subjects who were N-IgG positive had a PCR-confirmed infection before sample collection (Figure 1(B)), but this proportion increased over time. By Q2/2021 41% of N-IgG positive subjects had known of their infection by PCR test (Figure 1  (B)). During the second half of 2022, up to 78% of N-IgG positive subjects had knowledge of their infection either by a positive PCR test or at-home antigen test (Figure 1(B)).

Discussion
Here we show the development of immunity against SARS-CoV-2 in the Finnish adult population from the beginning of the COVID-19 pandemic to the end of 2022. Whilst S-IgG seroprevalence increased quickly in 2021 with the introduction of COVID-19 vaccinations, N-IgG seroprevalence remained below 7% until the last quartile of 2021. Following the emergence of Omicron variants, N-IgG seroprevalence increased rapidly and was 31% in Q1/2021 and 54% in Q4/2022 [14]. By the end of 2022, S-IgG seroprevalence was 99%, and 51% of our study population had vaccine and infection-induced antibodies, i.e. hybrid immunity.
The N-IgG seroprevalence estimate for 2020 may be an overestimation of all SARS-CoV-2 infections. When applied to low-prevalence populations, even for tests with high but imperfect specificity, the positive predictive value remains low, i.e. most positive test results are false positives [20]. On the contrary, later in the pandemic N-IgG seroprevalence may underestimate the number of SARS-CoV-2 infections due to the natural waning of antibody levels [21]. We have previously observed that the sensitivity of the N-IgG assay decreases after six months from infection [17,22]. By twelve months, N-IgG induced by past SARS-CoV-2 infections are difficult to distinguish from cross-reactive antibodies against seasonal coronaviruses [23,24] with our assay [15,22]. We have previously reported that the long-term sensitivity of the S-IgG assay is superior to N-IgG; S-IgG persisted in 97% of subjects at 13 months post-SARS-CoV-2 infection, whilst N-IgG were found in only 36% [15]. For this reason, N-IgG seroprevalence later in the pandemic is not representative of the cumulative number of infections, but rather an insight into more recent ones.
In 2020 most subjects had no antibody-mediated immunity to SARS-CoV-2 and we have previously shown that until June 2020, likely less than 1% of the Finnish population had been infected with SARS-CoV-2 [25]. One meta-analysis reported that nucleoprotein antibody seroprevalence increased steadily from 1.4% in March to 4.5% in December 2020 in European high-income countries. These estimates are very similar to the seroprevalences we observed in our study population. In 2021 most subjects had only vaccine-induced immunity despite the emergence and dominance of more transmissible Alpha [26,27] and Delta variants [14,28,29]. We observed that hybrid immunity levels remained low in 2021, indicating few breakthrough infections caused by these variants.
PCR testing capacity was initially limited in Finland [6,7] and our previous study shows that before April 2020 there were 4-17 infections (95% probability) for every PCR-confirmed infection [25]. Afterwards, the testing capacity increased, and by June 2020 there were 2-3 infections (50% probability) for every PCR-confirmed infection. Although there were some changes in COVID-19 testing policy during 2020 [6,10] and 2021 [11,12], our relatively low seroprevalence estimates indicate that the underreporting likely stayed at this modest level until the end of 2021.
In late-2021 PCR testing capacity was surpassed due to the sharp increase in cases caused by the Omicron variant, which was more contagious than previous variants and evaded previous immunity [30,31]. The limited access to PCR tests resulted in a change in testing guidelines [11,12], and at-home antigen tests became the primary diagnostic method in Finland. PCR tests were primarily offered to only those at risk of a severe infection [12] and laboratory-confirmed cases no longer represented the total disease burden. For this reason, serosurveillance became the best tool to follow the dynamics of the COVID-19 epidemic in Finland. We observed that the switch to the Omicron variant dominance in Finland [14] was followed by a 6% to 31% jump in N-IgG seroprevalence. Another study conducted in the greater-Helsinki area, Finland, observed very similar (28%) N antibody seroprevalence in March 2022 [32]. Additionally, a recent meta-analysis reported an average of 7% N antibody seroprevalence for high-income European countries in December 2021 and a rise to 48% by March 2022 [3], which is slightly higher than what we observed. Furthermore, breakthrough infections caused by the Omicron variants [31,33] are also apparent in our data; hybrid immunity rates increased from 4% to 31% between Q4/2021 and Q1/2022, and during this time there was a clear shift from only vaccine-induced immunity to hybrid immunity.
One key limitation of this study is that although we use a highly specific and sensitive S-IgG assay [17]  Dashed vertical lines divide calendar years. In 2020-2021 the oldest age group comprised of 65-70-year-olds and was extended to 65-85-year-olds in 2022. A. Development of SARS-CoV-2 nucleoprotein (N-IgG) and spike glycoprotein (S-IgG) seroprevalence in Finland by age group. Error bars represent 95% confidence intervals (Clopper-Pearson). Samples collected before Q3/2020 were not analysed for S-IgG. Timepoints with <10 samples per age group are not shown. B. Antibody-mediated of immunity by calendar year quartile. No immunity = N-IgG and S-IgG negative, unvaccinated before sampling. Only infection-induced immunity = Unvaccinated before sampling and N-IgG and/or S-IgG positive. Only vaccine-induced immunity = Had received at least one dose ≥14 days before sampling, S-IgG positive but N-IgG negative. Vaccinated without response = Had received at least one dose ≥14 days before sampling but were S-IgG negative. Hybrid immunity = Had received at least one dose ≥14 days before sampling and were N-IgG and S-IgG positive. Vaccinated S-IgG negative but N-IgG positive subjects (n = 2) were excluded from the categorization. Timepoints with <10 samples per age group are not shown. C. Number of samples in each age group and calendar year quartile.
which can detect antibodies for over a year after infection [15,22], spike antibodies cannot be used to assess SARS-CoV-2 infection-induced immunity after early 2021 due to mass COVID-19 vaccinations. Estimations of infection-induced antibody seroprevalence can thereafter only be based on N-IgG, which depicts only recent infections. It is also essential to highlight that the estimates of the prevalence of hybrid immunity presented here do not include information on cell-mediated immunity.
Another key limitation is the overall low participation rate (28%) and therefore possible selection bias, as the willingness to participate may be associated with the likelihood of a previous SARS-CoV-2 infection or COVID-19 vaccination. The participation rate was lower in younger age groups which we controlled for with age-adjustment. Population subgroups who are not native in either of Finland's two official languages were underrepresented in our study, which may have led to an underestimation of adult population immunity during the beginning of the epidemic, as COVID-19 incidence among these groups was initially higher in comparison to the native speakers. Half of the participants were from the capital region, which accounts for less than third of the total population of Finland. The capital region had initially the highest incidence of COVID-19 and we also observed higher seroprevalence there compared to other areas until the beginning of 2021. As individuals living in the capital region were overrepresented, we may have overestimated the seroprevalence in 2020 and the beginning of 2021. However, latest by 2022 the regional differences in seroprevalence had diminished and our results after that likely correspond well to the adult population in Finland.
The strengths of this study include the usage of well-validated and specific in-house antibody tests and population surveys spanning the Finnish adult population. Moreover, access to nationwide highquality records on PCR-confirmed infections and COVID-19 vaccinations provides valuable information for estimating assay sensitivity and adult population-wide hybrid immunity. By monitoring the seroprevalence over nearly three years, this study provides a longitudinal view of the COVID-19 epidemic and the evolution of immunity in the Finnish adult population. Previously published serosurveillance reports in Finland have been focused on shorter time periods [25,34], and seroprevalence in 2021 has not been reported elsewhere, making our study unique. Furthermore, at the time of writing this manuscript, we are among the first to report SARS-CoV-2 seroprevalence in late 2022 globally.
In this study, we have combined seroprevalence with data from the vaccination register and the infectious disease register and compared it to the genome sequencing data [14] of SARS-CoV-2 variants from the same period. This has allowed us to monitor population immunity in adults as vaccinations have progressed and during the dominance of different SARS-CoV-2 variants in Finland. Our results indicate that the containment measures in combination with high vaccine coverage were able to limit the spread of SARS-CoV-2 until the emergence of the highly transmissible Omicron variants. In 2022 the proportion of subjects with hybrid immunity increased, especially among the younger age groups, where more infections have occurred. Shortly after the start of the first Omicron wave, it was possible to observe the quick shift in the number of infections by serology, because most of the adult population were N-IgG negative before the wave and we were able to collect samples during the wave. To conclude, the findings of this study are significant for the understanding of the COVID-19 pandemic, and how different variants, containment measures and vaccinations affected the adult population. Furthermore, this study can be used to inform and improve strategies for preventing the spread of infectious diseases and mitigating the impacts of future outbreaks. in the clinical study design, Erika Lindh and Niko Tervo for providing SARS-CoV-2 variant surveillance data, the countless others who participated in sequencing and sample collection all over Finland and all participants of this study.

Disclosure statement
The Finnish Institute for Health and Welfare (THL) has until 9/2022 conducted Public-Private Partnerships with vaccine manufacturers and has previously received research funding for studies unrelated to COVID-19 from GlaxoS-

Funding
This study was funded by the Finnish Institute for Health and Welfare and the Academy of Finland (Decision number 336431).

Data availability
The data and code are available from the corresponding author upon reasonable request.